Post-translational covalent assembly of CAR and synNotch receptors for programmable antigen targeting

Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are engineered cell-surface receptors that sense a target antigen and respond by activating T cell receptor signaling or a customized gene program, respectively. Here, to expand the targeting capabilities of these receptors, we develop “universal” receptor systems for which receptor specificity can be directed post-translationally via covalent attachment of a co-administered antibody bearing a benzylguanine (BG) motif. A SNAPtag self-labeling enzyme is genetically fused to the receptor and reacts with BG-conjugated antibodies for covalent assembly, programming antigen recognition. We demonstrate that activation of SNAP-CAR and SNAP-synNotch receptors can be successfully targeted by clinically relevant BG-conjugated antibodies, including anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model. Finally, we develop a mathematical model to better define the parameters affecting universal receptor signaling. SNAP receptors provide a powerful strategy to post-translationally reprogram the targeting specificity of engineered cells.

1. The Notch system should be explained with more details. 2. The results section starts from the unsuccessful story of mSA2 binding to the biotinylated antibodies. It gives the story a negative tune. Will the authors consider moving this section to a later part of the article? This is an important finding that defines the binding threshold for a functional synNotch. Maybe if this section is moved to a later part, the article will flow better. 3. Figure 2a and 2b need more explanation, i.e. Gal4-VP64 is not explained. 4. The y-axis of 2e should be IL-7, instead of IL-17. 5. The kinetics of the antibody activation of the cells vary a lot in 2D. There should be some explanation/discussion on this. Does it mean the system needs optimization for each antibody? This will be very difficult to use in treatment settings. 6. In Figure 3, is it possible to add BFP data along with CD25 and CD62L? 7. In Figure 4C, are the cells gated on Snap-CAR+ cells? What is the CD4 vs CD8 ratio for the transduction? What are the memory phenotypes of the transduced cells? Suggest to add the information to supplementary. 8. In Figure 4, when using Fab Rituximab, was the Fab labelled with the same number of BG to the full-length antibody? 9. In Figure 5, it is not clear whether the effector cells are Jurkat cells or the human T cells. Human T cells should be used here and cell lysis assay should be performed. 10. In Figure 6b, Cetuximab CAR T cell panel, the blue line is missing. 11. Ref 38 on page 27's format is wrong. 12. Figure 4 legend, CD105a should be CD107a. 13. Figure S2C, +2 SNAPtag label should be moved down. 14. In Figure S2, why are the full-length antibodies only 55KDa? 15. In Figure S4, is it possible to change the table to curves? It is difficult to visualize the data. If it is not changed, please indicate the color intensity of the heatmap. 16. In Figure S5, T cell.Antibody line is missing.
Reviewer #2: Remarks to the Author: To expand the toolkit of parts available for modular receptor engineering, the authors engineered a "switchable" adaptor receptor system using post-translational, covalent attachment of an antibody to the extracellular domain of engineered receptors via SNAPtag technology. The novel aspects of this work include the use of a covalent labeling strategy with CARs and the extension of this adaptor approach to synNotch receptors, both of which would be of use to the field. While other adaptor-based CAR systems have been reported, this is the first adaptor synNotch.
However, there are several concerns that should be addressed: 1. Although the use of a covalent association strategy is a new approach to build adaptor receptor systems that have previously been constructed with weaker non-covalent interactions, the marginal impact of this approach is not entirely clear. Though an experimental comparison between the SNAPtag-based system and other existing adaptor systems is likely unnecessary, a model-based investigation could help to elucidate the regime in which covalent linkage would be advantageous. For example, the authors comment in the discussion that a scenario in which the antibody-target affinity is weaker could require a covalent adaptor system, but these concepts are only treated qualitatively. More quantitative analyses would help to define the impact of the SNAPtag platform on performance.
2. The use of the word "switchable" to describe this system is potentially misleading; "switchable" is often used to describe the ability of a system to move between 'on' and 'off' states, but that quality does not exist in this system. Additionally, this word can be taken to indicate general reversibility, which is also not necessarily possible given the covalent bond. Alternatively, consider using the word "modular" or "universal". 4. Page 4, paragraph 2: It would be useful to define the phrase "self-labeling". 5. Page 5, Results paragraph 1: references to Supplementary Figure 1 B & C are directed to the wrong panels within the figure and a reference to panel D is missing. Additionally, the stated Kd for msA1-biotin at the end of this paragraph seems erroneous; it should probably read 5.5x10^-9 as opposed to 10^9 M. 6. Page 6, paragraph 1: I believe that I understand what is meant by the hook effect (when antibody is in excess, saturation of target sites by free antibody competes with receptors for target engagement), but it would be worth clarifying for the reader. 7. Page 9, paragraph 1: it could be useful to state in the main text the technique used to quantify target cell lysis.
10. Page 10 in reference to the experiments in Figure 5a-b. It would be useful to comment briefly, at this point, as to whether the observed patterns were unexpected and perhaps to briefly speculate as to why patterns may differ between synNotch and CAR across these panels.
11. Figure  13. Figure 5b: Data points on the right (Raji) blue bar of the SNAP-synNotch panel are not scattered in the same way that the others are.
14. Figure 6b: It would be useful to indicate how these models were fit (i.e., Were the synNotch and CAR models corresponding to a single antibody fit simultaneously?). Additionally, please report all fitted parameters.

Response to Reviewers
We thank the reviewers for their positive comments and suggestions which have directed us to substantially improve the manuscript. We have addressed each of their remarks in the manuscript text as described below in bold.

Reviewer #1 (remarks to the author):
1. The in vitro responses are excellent, but there is no in vivo data provided. The trafficking of T cells to the tumors and the persistence of the CAR T cells is already a complicated process. In a more complicated system like this, the binding of antigen, antibody and receptors in vivo could be very difficult to predict. There should be data demonstrating that this system have some in vivo effect. In addition, the effect should be compared with a classic CAR. Answer: We agree with the reviewer about the importance of the in vivo data and now have now added the suggested in vivo mouse experiments to the manuscript. These new data include in vivo labeling of the SNAP-CAR T cells with to adaptor (Fig. 6) (Fig. 7 & Supplementary Fig. S11). This data includes a direct comparison to a classic CART targeting the same antigen (HER2).

Although this system provides flexibility for targeting cancers with different antigens, there is no data demonstrating this flexibility-i.e. a mixture of cancer cells with different antigen expressions. Answer: We have now added data showing the flexibility of the system to target a mixture of tumor cells with different antigen expressions (Fig. 4E). SNAP CAR T cells specifically lysed antigen+ tumor cells and adaptor combinations were able to elicit dual antigen targeting.
3. One of the advantages of the syn-Notch system is the capability to modify the output signals. However, in the syn-CAR system, the authors did not demonstrate this. It was shown in Figure 2 Jurkat cells produced IL-7. Is this replicable using the syn-CAR? If this is not possible, what is the difference between this syn-CAR, and a normal CAR binding to certain part of antibodies? Answer: We have revised the text to further clarify the synNotch system and its differences with the SNAP-CAR system. Briefly, the synNotch receptor has the sole output of producing a transgene while the SNAP-CAR activates T cell signaling receptor pathway. The production of a transgene in response to CAR activation in addition to activation of TCR signaling is possible by delivery of a NFAT promoter-driven construct which has been demonstrated by several other groups. The unique aspect of the synNotch system is the ability to deliver the desired transgene without activating other pathways.
Minor points: 1. The Notch system should be explained with more details. Answer: We have now added further explanation and description of the synNotch system in the Introduction and Results sections.
2. The results section starts from the unsuccessful story of mSA2 binding to the biotinylated antibodies. It gives the story a negative tune. Will the authors consider moving this section to a later part of the article? This is an important finding that defines the binding threshold for a functional synNotch. Maybe if this section is moved to a later part, the article will flow better. Answer: We agree with this comment and have now edited the text to shorten this section and move it to a later place in the paper. Figure 2a and 2b need more explanation, i.e. Gal4-VP64 is not explained. Answer: We have now added further explanation of the synNotch system which includes a description of the Gal4-VP64 part.

The y-axis of 2e should be IL-7, instead of IL-17.
Answer: We have corrected this in the figure.

The kinetics of the antibody activation of the cells vary a lot in 2D.
There should be some explanation/discussion on this. Does it mean the system needs optimization for each antibody? This will be very difficult to use in treatment settings.
Answer: We have added a discussion of this point into the Results and Discussion sections. In brief, the reviewer is correct that the curves are somewhat variable, and the system would likely require optimization in the clinic for a given adaptor. However, the qualitative behavior is still comparable showing universality and overall dosages that obtain peak activity are relatively similar across antibody/antigen pairs. It is likely that any approach that is being translated to the clinic will likely require dedicated focus and optimization. Figure 3, is it possible to add BFP data along with CD25 and CD62L? Answer: To clarify, while BFP is the output gene for the synNotch system in Fig 2., in Fig. 3 the BFP is a marker gene for CAR expression. For the activation marker analysis, CAR T cells in the co-incubation assay are identified by the BFP marker gene with activation genes CD62L and CD25 being quantified for these BFP+ cells. We updated the legend in the Fig. 3. to better explain the analysis. Figure 4C, are the cells gated on SNAP-CAR+ cells? What is the CD4 vs CD8 ratio for the transduction? What are the memory phenotypes of the transduced cells? Suggest to add the information to supplementary. Answer: Yes, the cells in Fig. 4C are gated on the SNAP-CAR+ population (BFP+). We have now included in the CD4 and CD8 flow cytometry data for the SNAP CAR T cells as well as the level of CD62L for these cells which informs on the effector/memory cell phenotype (Supplementary Fig. S9). These specific plots were characterizing the CAR T cells administered to mice (Fig. 7). Figure 4, when using Fab Rituximab, was the Fab labelled with the same number of BG to the full-length antibody? Answer: The average number of BG molecules were quantified and listed in Table#S1. The average number of BG molecules per full-length Rituximab antibody (2.8) was similar to the Fab (2.5).

In Figure 5, it is not clear whether the effector cells are Jurkat cells or the human T cells. Human T cells should be used here and cell lysis assay should be performed.
Answer: The results in Fig. 5. A, B were performed using Jurkat effector cells, however we have now added the requested cell lysis assays using primary human CAR T cells (Fig. 5C) Figure 6b, Cetuximab CAR T cell panel, the blue line is missing. Answer: In this figure the blue line is actually aligned with the orange curve so it is tricky to see, but it appears as a blue and orange dashed line.

Ref 38 on page 27's format is wrong.
Answer: We have corrected the formatting for this reference.
12. Figure 4 legend, CD105a should be CD107a. Answer: We have corrected the figure legend. Figure S2C, +2 SNAPtag label should be moved down. Answer: Due to the non-traditional covalent bond leading to branched protein chains the proteins run at a slightly higher than expected length in the denaturing gel, so the +2 SNAPtag label is at the correct size for what we have observed a single antibody chain and two SNAP proteins.

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14. In Figure S2, why are the full-length antibodies only 55KDa? Answer: The SDS-PAGE gels are denaturing gels, so one full length antibody molecule will be dissociated into 2 heavy chains of ~50kDa each and 2 light chains of ~25kDa each. Figure S4, is it possible to change the table to curves? It is difficult to visualize the data. If it is not changed, please indicate the color intensity of the heatmap. Answer: We have added a color key (now Supplementary Fig. S3) to make the heatmap easier to visualize and interpret. Figure S5, T cell.Antibody line is missing. Answer: This line is tricky to see, but it is there on the plot very close to the brown dashed line for the "T cell.Antibody.Tumor" population.

Reviewer #2 (Remarks to the Author): 1. Although the use of a covalent association strategy is a new approach to build adaptor receptor systems that have previously been constructed with weaker non-covalent interactions, the marginal impact of this approach is not entirely clear. Though an experimental comparison between the SNAPtag-based system and other existing adaptor systems is likely unnecessary, a model-based investigation could help to elucidate the regime in which covalent
linkage would be advantageous. For example, the authors comment in the discussion that a scenario in which the antibody-target affinity is weaker could require a covalent adaptor system, but these concepts are only treated qualitatively. More quantitative analyses would help to define the impact of the SNAPtag platform on performance. Answer: Based on our results comparing the mSA2 synNotch receptor with the SNAPtag receptor, a very high affinity (subnanomolar at least) attachment is likely required for the creation of an adaptor synNotch system (likely due to its unique receptor signaling mechanism of receptor pulling and proteolytic cleavage vs. receptor clustering for a CAR) making this covalent interaction paramount. For the adaptor CARs, we now include a lengthy discussion of the advantages of the covalent interaction in the discussion section. While unfortunately it is very difficult to comment on the exact affinity range that of adaptors in precise quantitative terms, we do see higher peak activation of the SNAP-CAR at lower adaptor doses compared to our previously published mSA2 adaptor CAR system. We now also mention this comparison in the text.
2. The use of the word "switchable" to describe this system is potentially misleading; "switchable" is often used to describe the ability of a system to move between 'on' and 'off' states, but that quality does not exist in this system. Additionally, this word can be taken to indicate general reversibility, which is also not necessarily possible given the covalent bond. Alternatively, consider using the word "modular" or "universal". Answer: We have removed the term "switchable" and have instead used "universal" or "adaptor" to describe the system. 4. Page 4, paragraph 2: It would be useful to define the phrase "self-labeling". Answer: We have now added text to define the term "self-labeling". Supplementary Figure 1 B & C are directed to the wrong panels within the figure and a reference to panel D is missing. Additionally, the stated Kd for msA1-biotin at the end of this paragraph seems erroneous; it should probably read 5.5x10^-9 as opposed to 10^9 M. Answer: We have now corrected these errors (Note: Supplementary Fig. 1 is now Supplementary Fig. 4).

Page 5, Results paragraph 1: references to
6. Page 6, paragraph 1: I believe that I understand what is meant by the hook effect (when antibody is in excess, saturation of target sites by free antibody competes with receptors for target engagement), but it would be worth clarifying for the reader. Answer: We have added a description of the hook effect to the text to be clarify this phenomenon. 7. Page 9, paragraph 1: it could be useful to state in the main text the technique used to quantify target cell lysis. Answer: We have now added a description to the main text to better describe this assay. Figure 6b: can you speculate as to why the Cetuximab+CAR T cells case was not improvable through parameter fitting? Answer: During parameter estimation, the literature values were used as initial estimates and bounded to one order of magnitude. With these constraints, we were able to minimize model error for seven of the antibody/receptor combinations. The eighth case (SNAP-CAR T cells with Cetuximab) was not able to converge which was potentially a result of the kinetic parameters being too far from the optimal values. We speculate that it is possible that the antibody binding to EGFR antigen was more affected by the presence of the randomly conjugated BG chemical motifs than the other antibodies thus leading to altered kinetics for the antibody adaptor vs. the un-modified Cetuximab antibody.

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9. Page 12, Discussion paragraph 1: "synNoch" should be "synNotch". Answer: We have corrected this typo. Figure 5a-b. It would be useful to comment briefly, at this point, as to whether the observed patterns were unexpected and perhaps to briefly speculate as to why patterns may differ between synNotch and CAR across these panels. Answer: We have included a discussion of the results including speculation on the differences of the synNotch and CAR constructs in the Results section. Figure 2d-e: Y-axes are incorrectly labeled IL-17 and should be labeled IL-7. Answer: We have corrected this error.

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12. Methods, Flow Cytometry Staining section: Was blocking of any sort used in surface staining procedures? Please describe if so. Answer: The FACS staining media contains 2% FBS which is noted, and no additional blocking is performed.
13. Figure 5b: Data points on the right (Raji) blue bar of the SNAP-synNotch panel are not scattered in the same way that the others are. Answer: This observed difference in data point scattering has to do with auto-formatting of how the GraphPad Prism software plots the data. For data points that are potentially overlapping, the software plots them horizontally, while data points that are not overlapping are plotted vertically. The Raji data points of the SNAP-synNotch panel have a higher level of variation and thus are plotted vertically by the software.
14. Figure 6b: It would be useful to indicate how these models were fit (i.e., Were the synNotch and CAR models corresponding to a single antibody fit simultaneously?). Additionally, please report all fitted parameters. Answer: The Methods section includes a description of the fitting process. Of note the synNotch and CAR models were fit separately, using the TagBFP MFI for synNotch (Fig. 2D) and CD25 MFI for the read-out of SNAP-CAR activation (Fig. 3D). We have now also included a table of the fitted parameter values (Table S5.)